[go: up one dir, main page]

WO2004048575A2 - Polypeptides de streptococcus pneumoniae - Google Patents

Polypeptides de streptococcus pneumoniae Download PDF

Info

Publication number
WO2004048575A2
WO2004048575A2 PCT/CA2003/001835 CA0301835W WO2004048575A2 WO 2004048575 A2 WO2004048575 A2 WO 2004048575A2 CA 0301835 W CA0301835 W CA 0301835W WO 2004048575 A2 WO2004048575 A2 WO 2004048575A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
fragments
analogs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CA2003/001835
Other languages
English (en)
Other versions
WO2004048575A3 (fr
Inventor
Josée Hamel
Nathalie Charland
Denis Martin
Bernard R. Brodeur
Normand Blais
Isabelle Pineau
Steve LABBÉ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shire Canada Inc
ID Biomedical Corp of Quebec
Original Assignee
Shire BioChem Inc
ID Biomedical Corp of Quebec
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shire BioChem Inc, ID Biomedical Corp of Quebec filed Critical Shire BioChem Inc
Priority to AU2003286056A priority Critical patent/AU2003286056A1/en
Publication of WO2004048575A2 publication Critical patent/WO2004048575A2/fr
Publication of WO2004048575A3 publication Critical patent/WO2004048575A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention is related to Streptococcus pneumoniae polypeptides and their corresponding DNA fragments, antigens, epitopes, antibodies directed to these epitopes, which may be used to prevent, diagnose and/or treat Streptococcus pneumoniae infections in mammals, such as humans .
  • S . pneumoniae is an important agent of disease in man especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia, septicaemia, pneumonia, meningitis with high morbidity and mortality throughout the world. Even with appropriate antibiotic therapy, pneumococcal infections still result in many deaths. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal disease, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S_-_ pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children.
  • Efforts to develop a pneumococcal vaccine have generally concentrated on generating immune responses to the pneumococcal capsular polysaccharide . More than 80 pneumococcal capsular serotypes have been identified on the basis on antigenic differences.
  • the currently available polysaccharide vaccine comprising 23 capsular polysaccharides that most frequently caused disease has significant shortcomings related primarily to the poor immunogenicity of many of the polysaccharides in infants, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups .
  • Phagocytosis and the subsequent killing of the ingested S . pneumoniae compose an immune mechanism that could clear the bacteria from infected individuals.
  • Bacterial surface antigens appear to be important targets for opsonic and protective antibody.
  • the use of a group-common immunogenic and protective pneumococcal surface protein antigen, either by itself or in combination with additional components, offers the possibility of a protein-based pneumococcal vaccine. This is the basis of our invention.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • polypeptides encoded by polynucleotides of the invention pharmaceutical compositions, vectors comprising polynucleotides of the invention operably linked to an expression control region, as well as host cells transfected with said vectors and processes for producing polypeptides comprising culturing said host cells under conditions suitable for expression.
  • Figure 1 represents the DNA sequence of SHB-SP-101 gene from SP64 strain; SEQ ID NO: 1
  • Figure 2 represents the amino acid sequence of SHB-SP-101 polypeptide from SP64 strain; SEQ ID NO: 2
  • Figure 3 represents the DNA sequence of SHB-SP-102 gene from
  • Figure 4 represents the amino acid sequence of SHB-SP-102 polypeptide from SP64 strain; SEQ ID NO: 4
  • Figure 5 represents the DNA sequence of SHB-SP-103 gene from
  • Figure 6 represents the amino acid sequence of SHB-SP-103 polypeptide from SP64 strain; SEQ ID NO: 6
  • Figure 7 represents the comparison of the amino acid sequences of SHB-SP-101 from ⁇ - pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • Figure 8 depicts the comparison of the amino acid sequences of SHB-SP-102 from S__ pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • Figure 9 depicts the comparison of the amino acid sequences of SHB-SP-103 from S ⁇ pneumoniae strains SP64, TIGR4 and R6 by using the program Align X from Vector NTI® sequence analysis software (version 7.0). Underneath the alignment, there is a consensus line where * and . characters indicate identical and similar amino acid residues, respectively.
  • Figure 10 represents the DNA sequence of SHB-SP-104 polypeptide; SEQ ID NO: 17.
  • Figure 11 epresents the amino acid sequence of SHB-SP-104 polypeptide; SEQ ID NO: 18.
  • the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5, 17 or fragments or analogs thereof;
  • the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:
  • a polynucleotide comprising a sequence chosen from SEQ ID No : 1, 3, 5 or 17;
  • the present invention provides an isolated polypeptide comprising a polypeptide chosen from:
  • polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No :
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • the present invention provides an isolated polypeptide comprising a polypeptide chosen from:
  • polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No :
  • polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • the present invention provides purified and isolated polynucleotides, which encode polypeptides which may be used to prevent, diagnose and/or treat Streptococcus pneumoniae infection.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs 5 thereof.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising a sequence 10 chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 1599% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention provides an 20 isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention provides an 25 isolated polynucleotide encoding a polypeptide having at least
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 3090% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 3595% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18. According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention provides an isolated polynucleotide encoding a polypeptide having at least 99% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • the present invention relates to polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention relates to polypeptides characterized by the amino acid sequence comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention relates to polypeptides characterized by the amino acid sequence comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18. According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • the present invention relates to epitope bearing portions of a polypeptide comprising a sequence chosen from SEQ ID No : 2 , 4, 6 or 18.
  • the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:
  • polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from: (a) a polynucleotide encoding a polypeptide having at least
  • the present invention provides an isolated polypeptide comprising a polypeptide chosen from: (a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof;
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof ;
  • polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; (g) the polypeptide of (a), (b) , (c) , (d) , (e) or (f) wherein the N-terminal Met residue is deleted; (h) the polypeptide of (a), (b) , (c) , (d) , (e) , (f) or (g) wherein the secretory amino acid sequence is deleted.
  • the present invention provides an isolated polypeptide comprising a polypeptide chosen from: (a) a polypeptide having at least 70% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18; (b) a polypeptide having at least 80% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide having at least 95% identity to a second polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • polypeptide capable of raising antibodies having binding specificity for a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18;
  • the invention includes DNA molecules, i.e. polynucleotides and their complementary sequences that encode analogs such as mutants, variants, homologues and derivatives of such polypeptides, as described herein in the present patent application.
  • the invention also includes RNA molecules corresponding to the DNA molecules of the invention.
  • the invention includes the corresponding polypeptides and monospecific antibodies that specifically bind to such polypeptides .
  • polypeptides in accordance with the present invention are antigenic.
  • polypeptides in accordance with the present invention are immunogenic.
  • polypeptides in accordance with the present invention can elicit an immune response in a host.
  • the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the polypeptides of the present invention as defined above.
  • An antibody that "has binding specificity” is an antibody that recognizes and binds the selected polypeptide but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes the selected peptide. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.
  • protection in the biological studies is defined by a significant increase in the survival curve, rate or period.
  • antigenic/immunogenic fragments of the polypeptides of the invention, or of analogs thereof are provided.
  • the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a polypeptide or analog thereof as described herein.
  • the present invention further provides fragments having at least 10 contiguous amino acid residues from the polypeptide sequences of the present invention. In one embodiment, at least 15 contiguous amino acid residues. In one embodiment, at least 20 contiguous amino acid residues. In one embodiment, at least 25 contiguous amino acid residues. In one embodiment, at least 30 contiguous amino acid residues. In one embodiment, at least 35 contiguous amino acid residues. In one embodiment, at least 40 contiguous amino acid residues.
  • fragments include those polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably conserved) and which may be natural or unnatural.
  • derivatives and analogs of polypeptides of the invention will have about 80% identity with those sequences illustrated in the figures or fragments thereof. That is, 80% of the residues are the same.
  • polypeptides will have greater than 80% identity.
  • polypeptides will have greater than 85% identity.
  • polypeptides will have greater than 90% identity.
  • polypeptides will have greater than 95% identity. In a further embodiment, polypeptides will have greater than 99% identity. In a further embodiment, analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10.
  • substitutions are those having a minimal influence on the secondary structure and hydropathic nature of the polypeptide.
  • substitutions are those known in the art as conserved, i.e. the substituted residues share physical or chemical properties such as hydrophobicity, size, charge or functional groups. These include substitutions such as those described by Dayhoff, M. in Atlas of Protein Sequence and
  • amino acids either natural or unnatural, belonging to one of the following groups represent conservative changes : ala, pro, gly, gin, asn, ser, thr, val; cys, ser, tyr, thr; val, ile, leu, met, ala, phe; lys, arg, orn, his; and phe, tyr, trp, his.
  • the preferred substitutions also include substitutions of D- enantiomers for the corresponding L-amino acids.
  • analogs or derivatives could be fusion polypeptides, incorporating moieties which render purification easier, for example by effectively tagging the desired polypeptide. It may be necessary to remove the "tag" or it may be the case that the fusion polypeptide itself retains sufficient antigenicity to be useful .
  • the percentage of homology is defined as the sum of the percentage of identity plus the percentage of similarity or conservation of amino acid type.
  • analogs of polypeptides of the invention will have about 70% homology with those sequences illustrated in the figures or fragments thereof.
  • polypeptides will have greater than 80% homology.
  • polypeptides will have greater than 85% homology.
  • polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10.
  • This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or homology for an optimal alignment.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
  • the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • what is important for analogs, derivatives and fragments is that they possess at least a degree of the antigenicity/ immunogenicity of the protein or polypeptide from which they are derived.
  • polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro- sequences; and (poly) saccharides .
  • PEG polyethylene glycol
  • amino acid regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different Streptococcus pneumoniae strains .
  • polypeptides of the present invention can be modified by terminal -NH 2 acylation (eg. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule .
  • terminal -NH 2 acylation eg. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine
  • hetero and homo polypeptide multimers of the polypeptide fragments and analogues include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or dimethylsuperimidate .
  • polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
  • the present invention also relates to chimeric polypeptides which comprise one or more polypeptides or fragments or analogs thereof as defined in the figures of the present application.
  • the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof; provided that the polypeptides are linked as to formed a chimeric polypeptide.
  • the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 provided that the polypeptides are linked as to formed a chimeric polypeptide.
  • a fragment, analog or derivative of a polypeptide of the invention will comprise at least one antigenic region i.e. at least one epitope.
  • polypeptides may be utilized having bishaloacetyl groups, nitroarylhalides, or the like, where the reagents being specific for thio groups. Therefore, the link between two mercapto groups of the different polypeptides may be a single bond or may be composed of a linking group of at least two, typically at least four, and not more than 16, but usually not more than about 14 carbon atoms.
  • polypeptide fragments and analogs of the invention do not contain a starting residue, such as methionine (Met) or valine (Val) .
  • polypeptides will not incorporate a leader or secretory sequence (signal sequence) .
  • the signal portion of a polypeptide of the invention may be determined according to established molecular biological techniques.
  • the polypeptide of interest may be isolated from a Streptococcus pneumoniae culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide.
  • polypeptides can be produced and/or used without their start codon (methionine or valine) and/or without their leader peptide to favor production and purification of recombinant polypeptides. It is known that cloning genes without sequences encoding leader peptides will restrict the polypeptides to the cytoplasm of E. coli and will facilitate their recovery (Glick, B.R. and Pasternak, J.J. (1998) Manipulation of gene expression in prokaryotes . In "Molecular biotechnology: Principles and applications of recombinant DNA", 2nd edition, ASM Press, Washington DC, p.109-143).
  • compositions of matter containing a polypeptide of the invention together with a carrier, diluent or adjuvant;
  • a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier, diluent or adjuvant;
  • a vaccine comprising a polypeptide of the invention and a pharmaceutically acceptable carrier, diluent or adjuvant;
  • a method for inducing an immune response against Streptococcus pneumoniae in a host, by administering to the host, an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to Streptococcus pneumoniae ; and particularly, (v) a method for preventing and/or treating a Streptococcus pneumoniae infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to a host in need
  • compositions of matter containing a polynucleotide of the invention together with a carrier, diluent or adjuvant;
  • a pharmaceutical composition comprising a polynucleotide of the invention and a carrier, diluent or adjuvant;
  • a method for inducing an immune response against Streptococcus pneumoniae in a host, by administering to the host, an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to Streptococcus pneumoniae; and particularly, (iv) a method for preventing and/or treating a Streptococcus pneumoniae infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to a host in need.
  • the polypeptides of the invention can also be coupled or conjugated to carrier proteins such as tetanus toxin, diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virus VP1 antigen or any other viral or bacterial toxin or antigen or any suitable proteins to stimulate the development of a stronger immune response.
  • carrier proteins such as tetanus toxin, diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virus VP1 antigen or any other viral or bacterial toxin or antigen or any suitable proteins to stimulate the development of a stronger immune response.
  • This coupling or conjugation can be done chemically or genetically.
  • a more detailed description of peptide-carrier conjugation is available in Van Regenmortel, M.H.V., Briand J.P., Muller S., Plaue S., «Synthetic Polypeptides as antigens» in Laboratory Techniques in Biochemistry and Molecular Biology
  • compositions comprising one or more Streptococcus pneumoniae polypeptides of the invention in a mixture with a pharmaceutically acceptable adjuvant.
  • Suitable adjuvants include (1) oil-in-water emulsion formulations such as MF59TM, SAFTM, RibiTM ; (2) Freund's complete or incomplete adjuvant; (3) salts i.e.
  • compositions of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or buccal or oral.
  • composition is also meant to include antibodies.
  • pharmaceutical composition is also meant to include antibodies.
  • one or more antibodies having binding specificity for the polypeptides of the present invention for the treatment or prophylaxis of Streptococcus pneumoniae infection and/or diseases and symptoms mediated by Streptococcus pneumoniae infection.
  • compositions of the invention are used for the prophylactic or therapeutic treatment of streptococcal infection and/or diseases and symptoms mediated by streptococcal infection as described in Manual of Clinical Microbiology, P.R. Murray (Ed, in chief),E.J. Baron, M.A. Pfaller, F.C. Tenover and R.H. Yolken. ASM Press, Washington, D.C. seventh edition, 1999, 1773p.
  • pharmaceutical compositions of the present invention are used for the prophylactic or therapeutic treatment of otitis media, sinusitis, pneumonia, meningitis, bacteremia, septicaemia.
  • compositions of the invention are used for the prophylactic or therapeutic treatment of streptococcal infection and/or diseases and symptoms mediated by streptococcal infection, in particular S.pneumoniae, group A streptococcus ( Streptococcus pyoqenes ) , group B streptococcus (GBS or Streptococcus aqalactiae) , Streptococcus dvsgalactiae. Streptococcus uberis , Streptococcus nocardia as well as Staphylococcus aureus .
  • the streptococcus infection is S.pneumoniae.
  • the invention provides a method for prophylactic or therapeutic treatment of Streptococcus pneumoniae infection in a host susceptible to Streptococcus pneumoniae infection comprising administering to said host a prophylactic or therapeutic amount of a composition of the invention.
  • the term "host" includes mammals.
  • the mammal is human.
  • the human is a neonate, infant or child.
  • the human is an adult.
  • compositions are administered to those hosts at risk of Streptococcus pneumoniae infection such as neonates, infants, children, elderly, immunocompromised hosts, hosts with an underlying disease that affect the innate mucosal immune system, such as chronic obstructive pulmonary disease and cystic fibrosis.
  • compositions of the invention are administered to adults.
  • compositions are preferably in unit dosage form of about 0.001 to 100 ⁇ g/kg (antigen/body weight) and more preferably 0.01 to 10 ⁇ g/kg and most preferably 0.1 to 1 ⁇ g/kg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • compositions are preferably in unit dosage form of about 0.1 ⁇ g to 10 mg and more preferably l ⁇ g to 1 mg and most preferably 10 to 100 ⁇ g 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
  • polypeptides characterized by the amino acid sequence comprising SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof .
  • polynucleotides are those illustrated in SEQ ID No: 1, 3, 5, 17 which may include the open reading frames (ORF) , encoding the polypeptides of the invention.
  • polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the polypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridize to the polynucleotide sequences herein above described (or the complement sequences thereof) having 70% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity. In a further embodiment, more than 98% identity. In a further embodiment, more than 99% identity.
  • Suitable stringent conditions for hybridation can be readily determined by one of skilled in the art (see for example Sambrook et al . , (1989) Molecular cloning : A Laboratory Manual, 2 nd ed, Cold Spring Harbor, N.Y. ; Current Protocols in Molecular Biology, (1999) Edited by Ausubel F.M. et al . , John Wiley 6- Sons, Inc., N.Y. ) .
  • the present invention provides polynucleotides that hybridize under stringent conditions to either
  • polypeptide comprises a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • the present invention provides polynucleotides that hybridize under stringent conditions to either
  • polypeptide comprises a sequence chosen from SEQ ID NO: 2, 4, 6 or 18.
  • present invention provides polynucleotides that hybridize under stringent conditions to either
  • polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6, 18 or fragments or analogs thereof.
  • the present invention provides polynucleotides that hybridize under stringent conditions to either
  • polypeptide comprises at least 10 contiguous amino acid residues from a polypeptide comprising a sequence chosen from SEQ ID No : 2, 4, 6 or 18.
  • polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5 or 17 or fragments or analogs thereof encoding polypeptides of the invention.
  • polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5 or 17 encoding polypeptides of the invention.
  • polynucleotides include both DNA and RNA.
  • the present invention also includes polynucleotides complementary to the polynucleotides described in the present application.
  • polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product.
  • the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation) .
  • the present invention provides host cells transfected with vectors comprising the polynucleotides of the invention.
  • host cells are transfected with vectors which encode the polypeptides of the invention, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.
  • Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non- chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA.
  • the polypeptide sequence may be incorporated in the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or Shine- Dalgarno sequence) , and optionally an operator (control element) .
  • an expression control region comprising a promoter, ribosome binding site (consensus region or Shine- Dalgarno sequence) , and optionally an operator (control element) .
  • Suitable promoters include but are not limited to LTR or SV40 promoter, E.coli lac, tac or trp promoters and the phage lambda P L promoter.
  • Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene.
  • Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pDlO phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNHl ⁇ a, PNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXTl , pSG, pSVK3, pBPV, pMSG and pSVL.
  • Host cells may be bacterial i.e.
  • E.coli Bacillus subtilis, Streptomyces ; fungal i.e. Asperqillus niqer, Asperqillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.
  • the present invention provides a process for producing a polypeptide comprising culturing a host cell of the invention under conditions suitable for expression of said polypeptide.
  • polypeptides Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC.
  • the polypeptides may be expressed with or without a leader or secretion sequence. In the former case the leader may be removed using post-translational processing (see US 4,431,739; US 4,425,437; and US 4,338,397) or be chemically removed subsequent to purifying the expressed polypeptide.
  • Streptococcus pneumoniae polypeptides of the invention may be used in a diagnostic test for Streptococcus pneumoniae infection.
  • a method for the detection of antibody specific to a Streptococcus pneumoniae antigen in a biological sample containing or suspected of containing said antibody may be performed as follows: a) obtaining a biological sample from a host; b) incubating one or more Streptococcus pneumoniae polypeptides of the invention or fragments thereof with the biological sample to form a mixture; and c) detecting specifically bound antigen or bound fragment in the mixture which indicates the presence of antibody specific to Streptococcus pneumoniae .
  • this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent assay (ELISA) , a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the protein are present in an organism.
  • an immunological test such as an enzyme-linked immunosorbent assay (ELISA)
  • ELISA enzyme-linked immunosorbent assay
  • radioimmunoassay or a latex agglutination assay
  • the DNA sequences encoding polypeptides of the invention may also be used to design DNA probes for use in detecting the presence of Streptococcus pneumoniae in a biological sample suspected of containing such bacteria.
  • the detection method of this invention comprises : a) obtaining the biological sample from a host; b) incubating one or more DNA probes having a DNA sequence encoding a polypeptide of the invention or fragments thereof with the biological sample to form a mixture; and c) detecting specifically bound DNA probe in the mixture which indicates the presence of Streptococcus pneumoniae bacteria.
  • the DNA probes of this invention may also be used for detecting circulating Streptococcus pneumoniae i.e. Streptococcus pneumoniae nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Streptococcus pneumoniae infections .
  • the probe may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labelled with a detectable label.
  • a preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention.
  • the preferred DNA probe will be an oligomer having a sequence complementary to at least about 15 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 30 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 50 contiguous nucleotides of the Streptococcus pneumoniae polypeptides of the invention.
  • Another diagnostic method for the detection of Streptococcus 5 pneumoniae in a host comprises : a) labelling an antibody reactive with a polypeptide of the invention or fragment thereof with a detectable label ; b) administering the labelled antibody or labelled 10 fragment to the host; and c) detecting specifically bound labelled antibody or labelled fragment in the host which indicates the presence of Streptococcus pneumoniae .
  • polynucleotides encoding polypeptides of the invention, or fragments, analogs or derivatives thereof, may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in
  • polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells.
  • the vector is injected intramuscularly.
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles [Wolf et al. H M G (1992) 1: 363, Turnes et al., Vaccine (1999), 17 : 2089, Le et al . , Vaccine (2000) 18 : 1893, Alves
  • a further aspect of the invention is the use of the Streptococcus pneumoniae polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of Streptococcus pneumoniae infection.
  • Suitable antibodies may be determined 5 using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against Streptococcus pneumoniae infection in a test model.
  • One example of an animal model is the mouse model described in the examples herein.
  • the antibody may be a whole antibody or 0 an antigen-binding fragment thereof and may belong to any immunoglobulm class.
  • the antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin.
  • the term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques .
  • the antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a 0 number of epitopes associated with the Streptococcus pneumoniae polypeptides but is preferably specific for one.
  • the present invention provides the use of an antibody for treatment and/or prophylaxis of 5 Streptococcus pneumoniae infections.
  • a further aspect of the invention is the use of the antibodies directed to the polypeptides of the invention for passive immunization.
  • a further aspect of the invention is a method for immunization, whereby an antibody raised by a polypeptide of the invention is administered to a host in an amount sufficient to provide a passive immunization.
  • the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the prophylactic or therapeutic treatment of Streptococcus pneumoniae infection.
  • the invention provides a kit comprising a polypeptide of the invention for detection or diagnosis of Streptococcus pneumoniae infection.
  • This example describes the amplification, cloning and expression of S_j_ pneumoniae gene fragments SHB-SP-101, SHB-SP- 102 and SHB-SP-103.
  • Gene fragments were amplified from genomic DNA isolated from S . pneumoniae serogroup 6 strain SP64 by PCR using pairs of oligonucleotide engineered to amplify the SHB-SP-101 (SEQ ID NO: 1), SHB-SP-102 (SEQ ID NO: 3) and SHB-SP-103 (SEQ ID NO: 5) DNA sequences.
  • Each of the primers had a restriction endonuclease site at the 5' end, thereby allowing directional in-frame cloning of the amplified product into the digested plasmid vector (see Table 1) .
  • PCR-amplified products were digested with restriction endonucleases and ligated to linearized plasmid pET19b (Novagen, Madison, WI ) expression vector.
  • pET plasmids were digested with restriction enzymes for the in-frame cloning. Clones were first stabilized in E_;_ coli DH5 ⁇ before introduction into E__ coli BL21( ⁇ DE3) or BL21 STAR ( ⁇ DE3) for expression of SHB-SP-101, SHB-SP-102 and SHB-SP-103 molecules .
  • Each of the resultant plasmid constructs was confirmed by nucleotide sequence analysis. The recombinant polypeptides were expressed as N-terminal fusions with His-tag.
  • the expressed recombinant polypeptides were purified from supernatant fractions obtained from centrifugation of sonicated IPTG-induced E_;_ coli cultures using a His-Bind metal chelation resin (QIAgen, Chatsworth, CA) .
  • the gene products generated are listed in the Table 2.
  • polypeptide sequences also contain a methionine starting residue.
  • EXAMPLE 2 This example describes the immunization of animals with recombinant gene products to elicit a protective immune response against pneumococcal disease.
  • mice Groups of 8 female BALB/c mice (Charles River, St-Constant, Canada) were immunized subcutaneously three times at three-week intervals with either 20 ⁇ g of SHB-SP-101, SHB-SP-102 or SHB- SP-103 polypeptides in presence of 10 ⁇ g of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada) or, as control, with QuilA adjuvant alone in PBS. Blood samples were collected from the orbital sinus on day 1, 22 and 43 prior to each immunization and seven days (day 50) following the third injection. Five to seven days later, the mice were challenged with approximately 10 5 CFU of the type 3 £___ pneumoniae strain 5 P4241.
  • This example describes the immunization of animals with a combination of recombinant gene products to elicit a protective immune response against pneumococcal disease.
  • This example illustrates the molecular conservation of 10 pneumococcal gene fragments .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des polypeptides de streptococcus et des polynucléotides codant ces polypeptides, des antigènes, des épitopes, des anticorps dirigés vers ces épitopes, qui peuvent être utilisés en vue de prévenir, de diagnostiquer et/ou de traiter des infections par Streptococcus pneumoniae chez des mammifères, tels que l'homme.
PCT/CA2003/001835 2002-11-26 2003-11-26 Polypeptides de streptococcus pneumoniae Ceased WO2004048575A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003286056A AU2003286056A1 (en) 2002-11-26 2003-11-26 Streptococcus pneumoniae surface polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US42891102P 2002-11-26 2002-11-26
US60/428,911 2002-11-26

Publications (2)

Publication Number Publication Date
WO2004048575A2 true WO2004048575A2 (fr) 2004-06-10
WO2004048575A3 WO2004048575A3 (fr) 2004-11-04

Family

ID=32393483

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2003/001835 Ceased WO2004048575A2 (fr) 2002-11-26 2003-11-26 Polypeptides de streptococcus pneumoniae

Country Status (2)

Country Link
AU (1) AU2003286056A1 (fr)
WO (1) WO2004048575A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2738577C1 (ru) * 2020-05-12 2020-12-14 Федеральное государственное бюджетное учреждение "Уральский научно-исследовательский институт охраны материнства и младенчества" Министерства здравоохранения Российской Федерации (ФГБУ "НИИ ОММ" Минздрава России) Способ раннего прогнозирования развития неонатальной пневмонии у недоношенных детей с экстремально низкой и очень низкой массой тела

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1770164B1 (fr) * 1996-10-31 2010-09-01 Human Genome Sciences, Inc. Antigènes et vaccins pour streptococcus pneumoniae
US6800744B1 (en) * 1997-07-02 2004-10-05 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
WO2000006738A2 (fr) * 1998-07-27 2000-02-10 Microbial Technics Limited Acides nucleiques et proteines de streptococcus pneumoniae
GB0107658D0 (en) * 2001-03-27 2001-05-16 Chiron Spa Streptococcus pneumoniae

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2738577C1 (ru) * 2020-05-12 2020-12-14 Федеральное государственное бюджетное учреждение "Уральский научно-исследовательский институт охраны материнства и младенчества" Министерства здравоохранения Российской Федерации (ФГБУ "НИИ ОММ" Минздрава России) Способ раннего прогнозирования развития неонатальной пневмонии у недоношенных детей с экстремально низкой и очень низкой массой тела

Also Published As

Publication number Publication date
WO2004048575A3 (fr) 2004-11-04
AU2003286056A1 (en) 2004-06-18
AU2003286056A8 (en) 2004-06-18

Similar Documents

Publication Publication Date Title
US7262024B2 (en) Streptococcus antigens
US20090186820A1 (en) Streptococcus pyogenes polypeptides and corresponding dna fragments
US8821895B2 (en) Streptococcus pyogenes antigens and corresponding DNA fragments
US7740870B2 (en) Group B Streptococcus antigens and corresponding DNA fragments
US20090074807A1 (en) Moraxella (branhamella) catarrhalis antigens
AU2002317107A1 (en) Group B streptococcus antigens and corresponding DNA fragments
US20080227706A1 (en) Bvh-a2 and bvh-a3 antigens of group b streptococcus
WO2004048575A2 (fr) Polypeptides de streptococcus pneumoniae
AU2002325109B2 (en) Moraxella (branhamella) catarrhalis polypeptides and corresponding DNA fragments
EP1399563B1 (fr) Antigenes de moraxella (branhamella) catarrhalis
AU2002302236A1 (en) Moraxella(Branhamella) catarrhalis antigens
WO2004007725A1 (fr) Polypeptide de streptococcus pyogenes
EP1390505A2 (fr) Antigenes du streptocoque de groupe b et fragments d'adn correspondants
AU2002325109A1 (en) Moraxella (branhamella) catarrhalis polypeptides and corresponding DNA fragments
AU2002344869A1 (en) Moraxella (branhamella) catarrhalis antigens

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP